Cytokines normally serve to enhance defense. However, when acting in excess, they may cause great damage, not lesser than that which pathogens can cause. In fact, in many diseases unwarranted effects of cytokines constitute a major pathogenic cause.
Cytokines of the TNF family regulate a wide range of different immune defense mechanisms, both of the innate and the adaptive types. Excessive function of several of them, including TNF (Genbank ID X01393) the Fas ligand (TNFSF6, Genbank ID U11821), CD40 ligand (TNFSF5, Genbank ID X67878) and others have been implicated in the pathology of various diseases. There is, in particular extensive evidence for a major pathological role of TNF in a wide range of diseases: infectious diseases such as malaria and sepsis, autoimmune diseases such as rheumatoid arthritis, the inflammatory bowel diseases and psoriasis, and certain types of cancer. Indeed, blocking TNF action by means such as anti-TNF antibodies or soluble TNF receptors was found to provide therapy at such situations [1] [2] [3].
In some pathological situations, including rheumatoid arthritis and Crohn's disease, a rather significant proportion of the patients respond favorably to anti-TNF therapy. There are, however, also patients with such diseases that respond rather poorly to these means, raising the need to define additional approaches for therapy [4].
Unlike many other cytokines that act solely as soluble proteins following their secretion by the cytokine-producing cell, the ligands of the TNF family are (with the exception of lymphotoxin (LTA, Genbank ID X01393) which is produced as a soluble secreted protein) produced as cell-bound type II transmembrane proteins. They can exert their effects in that form, affecting only cells that are located adjacently to the ligand-producing cell (juxtacrine regulation). Most of them are also shed, forming soluble molecules that circulate. Parts of those soluble ligands, for example TNF, are capable of acting as soluble cytokines, serving as paracrine regulators (affecting cells located relatively close to the ligand producing-cells) and endocrine regulators (affecting remote cells). Other ligands of the TNF family, for example the Fas ligand, do not act effectively in their shed form and may in that form even serve as antagonist to the cell-bound form [5] [6].
The occurrence of ligands of the TNF family on the surface of the cells producing them provides a potential means for specific targeting of these ligands producing cells. Such means can allow selective suppression or even elimination of the ligand producing cells at situations where the ligand plays a pathogenic role.
In several respects, destruction of cells producing a cytokine may turn to provide even better defense against the pathogenic effects of this cytokine than just direct blocking of the function of the cytokine molecules:
Destruction of the cytokine-producing cell prevents further synthesis of the cytokines and thus is likely to provide more durable protection than that obtained by just blocking the effect of the cytokine molecules that had been synthesized already.
Cells producing a cytokine often produce simultaneously some other cytokines that together serve to elicit a particular type of immune response. Well-known examples are the Th1- and Th2-type T lymphocytes, lymphocytes that produce distinct groups of cytokines, each serving to elicit a different type of immune defense [7]. Destroying cells producing a cytokine may thus, beside arrest of the synthesis of that particular cytokine, also result in arrest of synthesis of several other cytokines that assist the former in its pathogenic effects.
While blocking circulating cytokines affects the whole body, killing cytokine-producing cells can be restricted to a particular site in the body where these cells reside, thus allowing abolition of the cytokine deleterious effects at that particular site while maintaining beneficial effects of the cytokine at other sites.
Studies of the effect of anti-TNF therapy in Crohn's disease suggest that killing of TNF-producing cells may in some pathological situations indeed provide more effective therapy than that obtained by just blocking TNF. Therapeutic effects of anti-TNF antibodies in this disease were found to correlate with early induction of death of the TNF-producing cells by the antibodies [8] [9] [10]. There is thus a need to design means for effective and selective targeting of cells producing TNF or other ligands of the TNF family.
Cytotoxins joined to targeting molecules that bind to a cell-surface constituents can serve as potent cell-killing agents. Choosing a targeting moiety that recognizes a cell-type specific surface constituent can allow applying such cytotoxic chimera for selective destruction of specific cells in vivo. For example, chimeric fusion proteins comprised of antibodies against cancer-specific epitopes fused to Pseudomonas exotoxin (PE) or to Diphtheria toxin (DT) can specifically target and kill cancer cells. Such anti-cancer effects have also been obtained with chimera in which the toxins have been fused to ligands or hormones such as IL2, IL4 or IL13 whose receptors are prevalent in certain tumors. Likewise, cytotoxin-containing chimera were designed to be targeted to pathogen-afflicted cells. For example, HIV-infected cells can be selectively destroyed using immunotoxins comprised of an anti-gp120 antibody directed to the conserved CD4 binding site of gp120, or CD4, attached to a Pseudomonas exotoxin [1,1] [1,2] [13].
One kind of possible mean for targeting cytotoxins or other modulating agents to cells that express ligands of the TNF family is antibodies against these ligands. Indeed, antibodies against the CD40 ligand have been applied to target a toxin to CD40 ligand producing cells (Patent # EP1005372). However, only part of the antibodies produced against a particular ligand will bind effectively to the cell-bound form of this ligand, and of those—only part will be capable of competing with receptor molecules (soluble or cell-associated) once they bind to the ligand. Screening for such antibodies may turn to be highly involving and lengthy. Another disadvantage of antibodies, which are usually murine, is that they evoke an immune response in the patient.
It would, therefore, be desirable to define a general approach for generation of proteins that can target effectively cells producing ligands of the TNF family.